Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure.
Respiratory syncytial virus (RSV), a member of the paramyxovirus family, is the leading cause of viral pneumonia and bronchiolitis in infants and young children and is responsible for an estimated 95,000 hospitalizations and 4,500 deaths per year in the United States (IOM report, 1985; Hall and McBride, 1991; McIntosh and Chanock, 1990). Serious disease is most prevalent in infants 6 weeks to 6 months of age and in children with certain underlying illnesses (e.g. immunodeficiencies, congenital heart disease and bronchopulmonary dysplasia). Two major subgroups of RSV have been identified, A and B, as well as antigenic variants within each subgroup (Anderson et al, 1985; Mufson et al, 1985). Multiple variants of each subgroup have been found to cocirculate in epidemics which occur annually during late fall, winter, and spring months (Anderson et al, 1991). Most children are infected by 2 years of age. Complete immunity to RSV does not develop and reinfections occur throughout life (Henderson et al, 1979; Hall et al, 1991). Most infections are symptomatic and are generally confined to mild upper respiratory tract disease. A decrease in severity of disease is associated with two or more prior infections and, in some studies, with high levels of serum antibody, suggesting that protective immunity to RSV disease will accumulate following repeated infections (Lamprecht et al, 1976; Henderson et al, 1979; Glezen et al, 1981; Glezen et al, 1986; Kasel et al, 1987/88; Hall et al, 1991). There is also evidence that children infected with one of the two major RSV subgroups may be somewhat protected against reinfection with the homologous subgroup (Mufson et al, 1987). These observations suggest that it is both possible and worthwhile to develop an RSV vaccination regimen for infants and young children which would provide sufficient temporary immunity to protect against severe disease and death.
The identification of the two major subgroups of RSV has been based on reactivities of the F and G surface glycoproteins with monoclonal antibodies (Anderson et al., 1985; Mufson et al., 1985) and further delineated by sequence analysis (Collins, 1991; Sullender et al., 1991). Both F and G proteins elicit neutralizing antibodies and immunization with these proteins models (Johnson et al., 1987; Stott et al., 1987; Walsh et al., 1987). Most neutralizing antibodies are directed against the F protein. Beeler and Coelingh (1989) reported that out of 16 neutralization epitopes mapped to the F protein, 8 epitopes were conserved in all or all but one of 23 virus isolates tested. A high degree of sequence homology exists between the F protein of subgroups A and B (.about.90% amino acid and .about.80% nucleotide) whereas a much lower degree of homology exists between the G proteins (.about.50-60% amino acid and .about.60-70% nucleotide) (Collins, 1991). Correspondingly, immunity elicited by the F protein is more crossprotective between subgroups than is immunity elicited by the G protein (Johnson et al., 1987; Stott et al., 1987). In mice, humoral immunity induced by both the F and G proteins is thought to be responsible for protection against reinfection with virus (Connors et al., 1991) whereas the CTL response is thought to be more important in resolution of primary infections (Sun et al., 1983; Anderson et al., 1990; Graham et al., 1991). The 22K protein has been shown to be a potent inducer of cytotoxic lymphocytes (CTL) in mice, with lesser CTL recognition of F, N, and P proteins (Oppenshaw et al., 1990; Nicholas et al., 1990). Human CTL's have been shown to recognize the F, 22K, N, M, SH, and 1b proteins (Cherrie et al., 1992). This data suggests that the F proteins of either virus subgroup is a crucial immunogen in any RSV vaccine and that the G, 22K, N, M, SH, and 1b proteins should also be considered potential vaccine components. The benefit for vaccine efficacy in humans of using a live RSV vaccine or incorporating additional viral proteins into a subunit vaccine, and including viruses or proteins of both subgroups, remains to be elucidated.
Early attempts (1966) to vaccinate young children used a parenterally administered formalin-inactivated RSV vaccine. Unfortunately, administration of this vaccine in several field trials was shown to be specifically associated with the development of a significantly exacerbated illness following subsequent natural infection with RSV (Kapikian et al, 1969; Kim et al, 1969; Fulginiti et al, 1969; Chin et al; 1969). The reasons why this vaccine enhanced RSV disease are not clear. It has been suggested that this exposure to RSV antigen elicited an abnormal or unbalanced immune response which led to an immunopathological potentiation of natural disease (Kim et al, 1976; Prince et al, 1986). Following the lack of success with the formalin-inactivated vaccine, emphasis was placed on the development of live attenuated RSV vaccines. Vaccine candidates developed by cold adaptation were reduced in virulence in seropositive adults, however, one vaccine tested in seronegative infants was found to be under-attenuated (Kim et al, 1971; Forsyth and Phillips, 1973). RSV temperature sensitive (TS) mutants derived by chemical mutagenesis (Gharpure et al, 1969) were attenuated in rodent and non-human primate models (Wright et al, 1970; Richardson et al, 1978). Two mutants which initially appeared promising were found to be over- or under- attenuated in seronegative infants and to lack genetic stability (Kim et al, 1973; Hodes et al, 1974; McIntosh et al, 1974; Wright et al, 1976; Wright et al, 1982). Another vaccination approach using parenteral administration of live virus was found to lack efficacy and efforts along this line were discontinued (Belshe et al, 1982). Notably, these live RSV vaccines were never associated with disease enhancement as was the formalin-inactivated RSV vaccine.
Current RSV vaccine development efforts continue for both the live virus and subunit approaches. Because of previous experience with the formalin-inactivated RSV vaccine, trials of vaccines composed of non-replicating viral antigens have proceeded very cautiously. Only one subunit vaccine, purified F protein (PFP, Lederle-Praxis Biologicals) is currently in clinical trials. Studies in seropositive children have thus far given no indication of enhancement of natural disease. Other subunit vaccines in development include baculovirus produced chimeric FG protein (Brideau et al, 1989 Upjohn!; Wathen et al, 1991 Upjohn!) and peptides from F and G proteins (Trudel et al, 1991a,b). Vaccine approaches using live-attenuated RSV TS mutants (McKay et al, 1988; Watt et al, 1990) and recombinant vaccinia and adenoviruses expressing RSV F and G proteins (Olmstead et al, 1988; Collins et al, 1990a,b) are also being investigated. Use of a live-attenuated or live-vectored virus vaccine has several advantages over subunit or inactivated virus vaccines. An intranasally administered replicating virus will elicit systemic immunity. In addition, it is more likely than a parenterally administered subunit or inactivated vaccine to give a solid local mucosal immunity comprising both humoral and cellular components. This immunity may confer satisfactory protection from lower respiratory illness, as well as avoiding complications which could lead to enhanced disease.
Cold adaptation, a process by which virus is adapted to growth at temperatures colder than those at which it normally optimally grows, has been used to develop attenuated TS virus mutants for use as vaccines (for review see Maassab and DeBorde, 1985). This method generally results in the accumulation of multiple genetic lesions, unlike chemical mutagenesis in which the genetic lesions are usually single. These multiple lesions may help to confer phenotypic stability by reducing the probability that reversion of any one lesion will result in reversion of the relevant phenotype. Maassab has used stepwise cold adaptation to successfully develop several TS influenza vaccine candidates currently in clinical trials (Maassab et al, 1990; Obrosova-Serova et al, 1990; Edwards et al, 1991). These mutants, which bear attenuating mutations in at least four different genes, appear to be attenuated, immunogenic, and phenotypically stable. Belshe and co-workers have used cold adaptation to develop attenuated, TS strains of a paramyxovirus, parainfluenza virus type 3 (Belshe and Hissom, 1982; Murphy et al, 1990). In this case, cold adaptation was carried out in primary African green monkey kidney cells by reducing temperatures to 20.degree. C. Analysis of several virus variants cloned from this cold adapted population demonstrated that the level of attenuation and temperature sensitivity increased as the length of cold adaptation increased. These variants were shown to have reduced potential for virulence in humans, however the temperature sensitive phenotype was somewhat unstable in clinical trials (Clements et al, 1991). RSV was successfully cold adapted to 25-26.degree. C. in several laboratories in the mid 1960's, but was found to be under-attenuated in vaccine trials (Kim et al, 1971; Maassab and DeBorde, 1985; Forsyth and Phillips, 1973 (Lederle)). Maassab and DeBorde (1985) have suggested this may be because cold adaptation was not carried out at low enough temperatures, or clones of adequately attenuated virus were not isolated from a genetically mixed population of cold adapted virus.